WAPL induces cervical intraepithelial neoplasia modulated with estrogen signaling without HPV E6/E7.

Since cervical cancer still afflicts women around the world, it is necessary to understand the underlying mechanism of cervical cancer development. Infection with HPV is essential for the development of cervical intraepithelial neoplasia (CIN). In addition, estrogen receptor signaling is implicated in the development of cervical cancer. Previously, we have isolated human wings apart-like (WAPL), which is expected to cause chromosomal instability in the process of HPV-infected precancerous lesions to cervical cancer. However, the role of WAPL in the development of CIN is still unknown. In this study, in order to elucidate the role of WAPL in the early lesion, we established WAPL overexpressing mice (WAPL Tg mice) and HPV E6/E7 knock-in (KI) mice. WAPL Tg mice developed CIN lesion without HPV E6/E7. Interestingly, in WAPL Tg mice estrogen receptor 1 (ESR1) showed reduction as compared with the wild type, but cell growth factors MYC and Cyclin D1 controlled by ESR1 expressed at high levels. These results suggested that WAPL facilitates sensitivity of ESR1 mediated by some kind of molecule, and as a result, affects the expression of MYC and Cyclin D1 in cervical cancer cells. To detect such molecules, we performed microarray analysis of the uterine cervix in WAPL Tg mice, and focused MACROD1, a co-activator of ESR1. MACROD1 expression was increased in WAPL Tg mice compared with the wild type. In addition, knockdown of WAPL induced the downregulation of MACROD1, MYC, and Cyclin D1 but not ESR1 expression. Furthermore, ESR1 sensitivity assay showed lower activity in WAPL or MACROD1 downregulated cells than control cells. These data suggested that WAPL increases ESR1 sensitivity by activating MACROD1, and induces the expression of MYC and Cyclin D1. Therefore, we concluded that WAPL not only induces chromosomal instability in cervical cancer tumorigenesis, but also plays a key role in activating estrogen receptor signaling in early tumorigenesis.

Extracellular microvesicles that originated adipose tissue derived mesenchymal stem cells have the potential ability to improve rheumatoid arthritis on mice.

INTRODUCTION: Mesenchymal stem cells (MSCs) are promising therapeutic tools in regenerative medicine. In particularly adipose tissue derived MSC (AMSC) has powerful potential for the therapeutics of rheumatoid arthritis (RA) because these cells can control immune balance. RA systemically occurs autoimmune disease. Interestingly, IL-1 receptor antagonist deficient (IL-1ra-/-) mice induce inflammation in joints like RA. In RA therapy, although AMSC improves the inflammation activity, it is little known to play roles of extracellular microvesicles (EV) for improvement of RA. To clarify the MSC-derived EVs are involved amelioration mechanisms for RA by themselves, we examined the functional effects of development for RA by AMSC-EVs. METHODS: We isolated AMSCs derived mice adipose tissue and purified EVs from the culture supernatant of AMSCs. To examine whether EVs can improve RA, we administrated EVs or AMSCs to IL-1ra knockout mice as RA model mice. We analyzed EVs-included factor by western blot methods and RA improvement effect by ELISA. RESULTS: In this study, we showed that the swellings of joints on mice in wild type AMSC and that in AMSC-EVs decreased than that in IL-1ra-/- mice-AMSC-EVs and in none-treated. We detected IL-1ra expression in AMSC-EVs in wild type mice but not that in IL-1ra-/- mice. Proinflammatory cytokine expression changes in mice showed in AMSCs and AMSC-EVs, but no apparent differences cytokine expressions were detected in IL-1ra-/- mice. CONCLUSIONS: In this study, we concluded that MSCs might improve RA by the transferring of factors such as IL-1ra, which are included their MSC derived- EVs.

An improved Red/ET recombineering system and mouse ES cells culture conditions for the generation of targeted mutant mice.

Targeted mutant mice generated on a C57BL/6 background are powerful tools for analysis of the biological functions of genes, and gene targeting technologies using mouse embryonic stem (ES) cells have been used to generate such mice. Recently, a bacterial artificial chromosome (BAC) recombineering system was established for the construction of targeting vectors. However, gene retrieval from BACs for the generation of gene targeting vectors using this system remains difficult. Even when construction of a gene targeting vector is successful, the efficiency of production of targeted mutant mice from ES cells derived from C57BL/6 mice are poor. Therefore, in this study, we first improved the strategy for the retrieval of genes from BACs and their transfer into a DT-A plasmid, for the generation of gene targeting vectors using the BAC recombineering system. Then, we attempted to generate targeted mutant mice from ES cell lines derived from C57BL/6 mice, by culturing in serum-free medium. In conclusion, we established an improved strategy for the efficient generation of targeted mutant mice on a C57BL/6 background, which are useful for the in vivo analysis of gene functions and regulation.

Differential hepatic distribution of insulin receptor substrates causes selective insulin resistance in diabetes and obesity.

Hepatic insulin signalling involves insulin receptor substrates (Irs) 1/2, and is normally associated with the inhibition of gluconeogenesis and activation of lipogenesis. In diabetes and obesity, insulin no longer suppresses hepatic gluconeogenesis, while continuing to activate lipogenesis, a state referred to as 'selective insulin resistance'. Here, we show that 'selective insulin resistance' is caused by the differential expression of Irs1 and Irs2 in different zones of the liver. We demonstrate that hepatic Irs2-knockout mice develop 'selective insulin resistance', whereas mice lacking in Irs1, or both Irs1 and Irs2, develop 'total insulin resistance'. In obese diabetic mice, Irs1/2-mediated insulin signalling is impaired in the periportal zone, which is the primary site of gluconeogenesis, but enhanced in the perivenous zone, which is the primary site of lipogenesis. While hyperinsulinaemia reduces Irs2 expression in both the periportal and perivenous zones, Irs1 expression, which is predominantly in the perivenous zone, remains mostly unaffected. These data suggest that 'selective insulin resistance' is induced by the differential distribution, and alterations of hepatic Irs1 and Irs2 expression.

Development of Novel Small Hairpin RNAs That do not Require Processing by Dicer or AGO2.

The innate cytokine response to nucleic acid is the most challenging problem confronting the practical use of nucleic acid medicine. The degree of stimulation of the innate cytokine response strongly depends on the length of the nucleic acid. In this study, we developed a 30-nucleotide single-strand RNA, termed "guide hairpin RNA (ghRNA, ghR)", that has a physiological function similar to that of miRNA and siRNA. The ghR caused no innate cytokine response either in vitro or in vivo. In addition, its structure does not contain a passenger strand seed sequence, reducing the unwanted gene repression relative to existing short RNA reagents. Systemic and local injection of ghR-form miR-34a (ghR-34a) suppressed tumor growth in a mouse model of RAS-induced lung cancer. Furthermore, Dicer and AGO2 are not required for ghR-34a function. This novel RNA interference (RNAi) technology may provide a novel, safe, and effective nucleic acid drug platform that will increase the clinical usefulness of nucleic acid therapy.

TCF7L2 in mouse pancreatic beta cells plays a crucial role in glucose homeostasis by regulating beta cell mass.

AIMS/HYPOTHESIS: Common genetic variations of the transcription factor 7-like 2 gene (encoded by TCF7L2), one of the T cell factor/lymphoid enhancer-binding factor transcription factors for the converging wingless-type MMTV integration site family (Wnt)/ß-catenin signalling pathway, are known to be associated with type 2 diabetes. Individuals with at-risk alleles of TCF7L2 exhibit impaired insulin secretion. Although previous studies using animal models have revealed the existence of a relationship between the Wnt/ß-catenin signalling pathway and glucose homeostasis, it remains unclear whether TCF7L2 in the pancreatic beta cells might be causally involved in insulin secretion in vivo. In this study, we investigated the role of TCF7L2 expressed in the pancreatic beta cells in glucose homeostasis. METHODS: Three independent groups of genetically engineered mice (DN mice) were generated, in which expression of the dominant-negative form of Tcf7l2 was driven under a rat insulin promoter. Phenotypes of both adult and newborn mice were evaluated. The levels of genes and proteins expressed in isolated islets were determined by reverse transcription-quantitative PCR and western blot analysis, respectively. RESULTS: Adult DN mice showed impaired glucose tolerance and decreased insulin secretion in both oral and intraperitoneal glucose tolerance tests. Marked reduction of the beta cell area and whole-pancreas insulin content was observed in both the adult and newborn DN mice. Islets from the DN mice showed decreased gene expressions of Ccnd1, Ccnd2, Irs1, Irs2, Ins1, Ins2 and Mafa, consistent with the deleterious effects of the dominant-negative form of Tcf7l2 on beta cell proliferation and insulin production. CONCLUSIONS/INTERPRETATION: TCF7L2 expressed in the pancreatic beta cells plays a crucial role in glucose metabolism through regulation of the beta cell mass.

Evi1 is essential for hematopoietic stem cell self-renewal, and its expression marks hematopoietic cells with long-term multilineage repopulating activity.

Ecotropic viral integration site 1 (Evi1), a transcription factor of the SET/PR domain protein family, is essential for the maintenance of hematopoietic stem cells (HSCs) in mice and is overexpressed in several myeloid malignancies. Here, we generate reporter mice in which an internal ribosome entry site (IRES)-GFP cassette is knocked-in to the Evi1 locus. Using these mice, we find that Evi1 is predominantly expressed in long-term HSCs (LT-HSCs) in adult bone marrow, and in the hematopoietic stem/progenitor fraction in the aorta-gonad-mesonephros, placenta, and fetal liver of embryos. In both fetal and adult hematopoietic systems, Evi1 expression marks cells with long-term multilineage repopulating activity. When combined with conventional HSC surface markers, sorting according to Evi1 expression markedly enhances purification of cells with HSC activity. Evi1 heterozygosity leads to marked impairment of the self-renewal capacity of LT-HSCs, whereas overexpression of Evi1 suppresses differentiation and boosts self-renewal activity. Reintroduction of Evi1, but not Mds1-Evi1, rescues the HSC defects caused by Evi1 heterozygosity. Thus, in addition to documenting a specific relationship between Evi1 expression and HSC self-renewal activity, these findings highlight the utility of Evi1-IRES-GFP reporter mice for the identification and sorting of functional HSCs.

Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle.

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.

Generation of transgenic mice on an NOD/SCID background using the conventional microinjection technique.

Humanized mice, which refers to immunodeficient mice repopulated with the human immune system, are powerful tools for study in the field of immunology. It has been difficult, however, to generate these transgenic (Tg) mice directly from such strains as the NOD/SCID mouse. In this study, we describe a method developed by us for the generation of Tg mice on an NOD/SCID background. First, we obtained fertilized eggs efficiently by means of in vitro fertilization (IVF); then, we attempted to generate CAG-EGFP Tg mice on an NOD/SCID background, finding that delayed timing of the microinjection after the IVF improved the time to development of the two-cell-stage embryos and the obtainment of newborns. We successfully generated Tg mice and confirmed the germ-line transmission in the offspring. In conclusion, we established a novel system for directly generating transgenic mice on an NOD/SCID background. This novel system is expected to allow improved efficiency of the generation of humanized mice.

Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions.

Adiponectin plays a central role as an antidiabetic and antiatherogenic adipokine. AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro, and their reduction in obesity seems to be correlated with reduced adiponectin sensitivity. Here we show that adenovirus-mediated expression of AdipoR1 and R2 in the liver of Lepr(-/-) mice increased AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor (PPAR)-alpha signaling pathways, respectively. Activation of AMPK reduced gluconeogenesis, whereas expression of the receptors in both cases increased fatty acid oxidation and lead to an amelioration of diabetes. Alternatively, targeted disruption of AdipoR1 resulted in the abrogation of adiponectin-induced AMPK activation, whereas that of AdipoR2 resulted in decreased activity of PPAR-alpha signaling pathways. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions, resulting in increased tissue triglyceride content, inflammation and oxidative stress, and thus leading to insulin resistance and marked glucose intolerance. Therefore, AdipoR1 and R2 serve as the predominant receptors for adiponectin in vivo and play important roles in the regulation of glucose and lipid metabolism, inflammation and oxidative stress in vivo.

Overexpression of monocyte chemoattractant protein-1 in adipose tissues causes macrophage recruitment and insulin resistance.

Adipose tissue expression and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) correlate positively with adiposity. To ascertain the roles of MCP-1 overexpression in adipose, we generated transgenic mice by utilizing the adipocyte P2 (aP2) promoter (aP2-MCP-1 mice). These mice had higher plasma MCP-1 concentrations and increased macrophage accumulation in adipose tissues, as confirmed by immunochemical, flow cytometric, and gene expression analyses. Tumor necrosis factor-alpha and interleukin-6 mRNA levels in white adipose tissue and plasma non-esterified fatty acid levels were increased in transgenic mice. aP2-MCP-1 mice showed insulin resistance, suggesting that inflammatory changes in adipose tissues may be involved in the development of insulin resistance. Insulin resistance in aP2-MCP-1 mice was confirmed by hyperinsulinemic euglycemic clamp studies showing that transgenic mice had lower rates of glucose disappearance and higher endogenous glucose production than wild-type mice. Consistent with this, insulin-induced phosphorylations of Akt were significantly decreased in both skeletal muscles and livers of aP2-MCP-1 mice. MCP-1 pretreatment of isolated skeletal muscle blunted insulin-stimulated glucose uptake, which was partially restored by treatment with the MEK inhibitor U0126, suggesting that circulating MCP-1 may contribute to insulin resistance in aP2-MCP-1 mice. We concluded that both paracrine and endocrine effects of MCP-1 may contribute to the development of insulin resistance in aP2-MCP-1 mice.